Abstract
The metabolism of glutamate into ornithine, arginine, proline, and polyamines is a major network of nitrogen-metabolizing pathways in plants, which also produces intermediates like nitric oxide, and γ-aminobutyric acid (GABA) that play critical roles in plant development and stress. While the accumulations of intermediates and the products of this network depend primarily on nitrogen assimilation, the overall regulation of the interacting sub-pathways is not well understood. We tested the hypothesis that diversion of ornithine into polyamine biosynthesis (by transgenic approach) not only plays a role in regulating its own biosynthesis from glutamate but also affects arginine and proline biosynthesis. Using two high putrescine producing lines of Arabidopsis thaliana (containing a transgenic mouse ornithine decarboxylase gene), we studied the: (1) effects of exogenous supply of carbon and nitrogen on polyamines and pools of soluble amino acids; and, (2) expression of genes encoding key enzymes in the interactive pathways of arginine, proline and GABA biosynthesis as well as the catabolism of polyamines. Our findings suggest that: (1) the overall conversion of glutamate to arginine and polyamines is enhanced by increased utilization of ornithine for polyamine biosynthesis by the transgene product; (2) proline and arginine biosynthesis are regulated independently of polyamines and GABA biosynthesis; (3) the expression of most genes (28 that were studied) that encode enzymes of the interacting sub-pathways of arginine and GABA biosynthesis does not change even though overall biosynthesis of Orn from glutamate is increased several fold; and (4) increased polyamine biosynthesis results in increased assimilation of both nitrogen and carbon by the cells.
Highlights
The glutamate (Glu) to proline (Pro), ornithine (Orn), arginine (Arg), polyamines (PAs), and γ-aminobutyric acid (GABA) group of reactions constitutes one of the major interactive pathways for carbon (C) and nitrogen (N) assimilation and partitioning (Figure 1)
We report here the results of our study involving: (1) the effects of supplementary N and C application on cellular PAs and amino acids in the wild type (WT) and transgenic plants of Arabidopsis thaliana, which produce several-fold higher concentrations of Put via the constitutive (2x35S CaMV promoter) expression of mouse ODC (mODC) gene; and (2) the effects of inducible expression of mODC on the expression of genes encoding several enzymes of the Orn→Arg, Orn⇔Pro, Glu/PAs→GABA pathways, as well as those involved with initial steps in the PA catabolism pathway
Since Orn is a limiting metabolite for continued production of Put by the transgenic mODC, and since it is present in relatively small concentrations, and is largely produced from Glu whose biosynthesis requires the continued supply/assimilation of N and C, two specific questions were addressed: (1) Will increased supply of N or C in the growth medium allow extra Put to be produced in the WT and/or the transgenic plants? (2) Will the increased availability of either N or C in the medium affect the amino acids pool in the WT and/or the transgenic plants?
Summary
The glutamate (Glu) to proline (Pro), ornithine (Orn), arginine (Arg), polyamines (PAs), and γ-aminobutyric acid (GABA) group of reactions constitutes one of the major interactive pathways for carbon (C) and nitrogen (N) assimilation and partitioning (Figure 1). The products of this pathway have a wide range of physiological functions in plants. The three PAs are obligatory requirements for cell survival and growth through their molecular interactions with nucleic acids (transcription and translation) and cellular membranes (Kusano et al, 2007; Minocha et al, 2014); they are a source of GABA, which plays critical roles in diverse cellular functions in plants (Shelp et al, 2012a). Further complexity of cellular PA functions involves their interactions with plasma membrane cellular H+ pumps (Garufi et al, 2007) and the transport of Ca2+ and K+ across root membranes in a species-specific manner (Zepeda-Jazo et al, 2011)
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